Characterization of a human thyroid microtissue model for testing thyroid disrupting chemicals.

Perturbation of thyroid hormone (T4) synthesis is known to cause numerous developmental, metabolic, and cognitive disorders in humans. Due to species differences in sensitivity to chemical exposures, there is a need for human-based in vitro approaches that recapitulate thyroid cellular architecture and T4 production when screening. To address these limitations, primary human thyrocytes, isolated from healthy adult donor tissues and cryopreserved at passage one (p'1) were characterized for cellular composition, 3D follicular architecture, and thyroglobulin (TG)/T4 expression and inhibition by prototype thyroid disrupting chemicals (TDC). Flow analysis of the post-thaw cell suspension showed >80% EpCAM-positive cells with 10%-50% CD90-positive cells. When seeded onto 96-well Matrigel®-coated plates and treated with bovine thyroid stimulating hormone (TSH), thyrocytes formed 3D microtissues during the initial 4-5 days of culture. The microtissues exhibited a stable morphology and size over a 14-day culture period. TG and T4 production were highest in microtissues when the proportion of CD90-positive cells, seeding density and thyroid stimulating hormone concentrations were between 10%-30%, 6K-12K cells per well, and 0.03-1 mIU/mL, respectively. At maximal TG and T4 production levels, average microtissue diameters ranged between 50 and 200 µm. The T4 IC50 values for two prototype TPO inhibitors, 6-propyl-2-thiouracil and methimazole, were ∼0.7 µM and ∼0.5 µM, respectively, in microtissue cultures treated between days 9 and 14. Overall, p'1 cryopreserved primary human thyrocytes in 3D microtissue culture represent a promising new model system to prioritize potential TDC acting directly on the thyroid as part of a weight-of-evidence hazard characterization.

Bioprinting on sheet-based scaffolds applied to the creation of implantable tissue-engineered constructs with potentially diverse clinical applications: Tissue-Engineered Muscle Repair (TEMR) as a representative testbed.

Purpose: This report explores the overlooked potential of bioprinting to automate biomanufacturing of simple tissue structures, such as the uniform deposition of (mono)layers of progenitor cells on sheetlike decellularized extracellular matrices (dECM). In this scenario, dECM serves as a biodegradable celldelivery matrix to provide enhanced regenerative microenvironments for tissue repair. The Tissue-Engineered Muscle Repair (TEMR) technology-where muscle progenitor cells are seeded onto a porcine bladder acellular matrix (BAM), serves as a representative testbed for bioprinting applications. Previous work demonstrated that TEMR implantation improved functional outcomes following VML injury in biologically relevant rodent models.Materials and Methods: In the described bioprinting system, a cell-laden hydrogel bioink is used to deposit high cell densities (1.4 × 105-3.5 × 105 cells/cm2), onto both sides of the bladder acellular matrix as proof-of-concept.Results: These bioprinting methods achieve a reproducible and homogeneous distribution of cells, on both sides of the BAM scaffold, after just 24hrs, with cell viability as high as 98%. These preliminary results suggest bioprinting allows for improved dual-sided cell coverage compared to manual-seeding.Conclusions: Bioprinting can enable automated fabrication of TEMR constructs with high fidelity and scalability, while reducing biomanufacturing costs and timelines. Such bioprinting applications are underappreciated, yet critical, to expand the overall biomanufacturing paradigm for tissue engineered medical products. In addition, biofabrication of sheet-like implantable constructs, with cells deposited on both sides, is a process that is both scaffold and cell-type agnostic, and furthermore, is amenable to many geometries, and thus, additional tissue engineering applications beyond skeletal muscle.

Liver-Regenerative Transplantation: Regrow and Reset.

Liver transplantation, either a partial liver from a living or deceased donor or a whole liver from a deceased donor, is the only curative therapy for severe end-stage liver disease. Only one-third of those on the liver transplant waiting list will be transplanted, and the demand for livers is projected to increase 23% in the next 20 years. Consequently, organ availability is an absolute constraint on the number of liver transplants that can be performed. Regenerative therapies aim to enhance liver tissue repair and regeneration by any means available (cell repopulation, tissue engineering, biomaterials, proteins, small molecules, and genes). Recent experimental work suggests that liver repopulation and engineered liver tissue are best suited to the task if an unlimited availability of functional induced pluripotent stem (iPS)-derived liver cells can be achieved. The derivation of iPS cells by reprogramming cell fate has opened up new lines of investigation, for instance, the generation of iPS-derived xenogeneic organs or the possibility of simply inducing the liver to reprogram its own hepatocyte function after injury. We reviewed current knowledge about liver repopulation, generation of engineered livers and reprogramming of liver function. We also discussed the numerous barriers that have to be overcome for clinical implementation.

Establishment of short-term primary human prostate xenografts for the study of prostate biology and cancer.

Human tissue xenograft models are currently the only tool for conducting in vivo analyses of intact human tissue. The goal of the present study was to develop reliable methods for successful generation of short-term primary tissue xenografts from benign and tumor-derived human prostate tissue. Primary human prostate xenografts were established in athymic nu/nu mice from eight of eight benign and five of five prostate cancer tissues, collected from a total of 10 patients who underwent radical prostatectomy for the treatment of prostate cancer. An average of 13 xenografts was established per specimen. Two tissue specimens were cryopreserved for >1 month before successful generation of prostate xenografts. After 1 month in vivo, xenograft tissues were harvested and examined regarding: gross evidence of vascularization; tissue morphology; proliferation; apoptosis; and expression of androgen receptor, prostate-specific antigen, and high molecular weight cytokeratins specific for basal cells in the prostate. Direct comparison of the original tissue specimen and the 1-month xenografts revealed similar histology; similar apoptotic and proliferative fractions in most cases; and comparable expression levels and expression patterns of androgen receptor, prostate-specific antigen, and high molecular weight cytokeratins. These data demonstrate that primary human prostate xenografts, benign and malignant, can be established routinely from human prostate tissue surgical specimens, and that the xenografts maintain tissue architecture and expression of key prostatic markers. The development of this methodology, including the technique for cryopreservation of human tissue, will allow multiple (successive) analyses of human prostate tissue to be conducted throughout time using a tissue sample derived from a single patient; and simultaneous analysis of human prostate tissues derived from a cohort of patients.

Aberrant cell cycle checkpoint function in transformed hepatocytes and WB-F344 hepatic epithelial stem-like cells.

Cell cycle checkpoints are barriers to carcinogenesis as they function to maintain genomic integrity. Attenuation or ablation of checkpoint function may enhance tumor formation by permitting outgrowth of unstable cells with damaged DNA. To examine the function of cell cycle checkpoints in rat hepatocarcinogenesis, we analyzed the responses of the G (1), G (2) and mitotic spindle assembly checkpoints in normal rat hepatocytes, hepatic epithelial stem-like cells (WB-F344) and transformed derivatives of both. Normal rat hepatocytes (NRH) displayed a 73% reduction in the fraction of nuclei in early S-phase 6-8 h following 8 Gy of ionizing radiation (IR) as a quantitative measure of G (1) checkpoint function. Chemically and virally transformed hepatocyte lines displayed significant attenuation of G (1) checkpoint function, ranging from partial to complete ablation. WB-F344 rat hepatic epithelial cell lines at low, mid and high passage levels expressed G (1) checkpoint function comparable with NRH. Only one of four malignantly transformed WB-F344 cell lines displayed significant attenuation of G (1) checkpoint function. Attenuation of G (1) checkpoint function in transformed hepatocytes and WB-F344 cells was associated with alterations in p53, ablated/attenuated induction of p21 (Waf1) by IR, as well as aberrant function of the spindle assembly checkpoint. NRH displayed 93% inhibition of mitosis 2 h after 1 Gy IR as a quantitative measure of G (2) checkpoint function. All transformed hepatocyte and WB-F344 cell lines displayed significant attenuation of the G (2) checkpoint. Moreover, the parental WB-F344 line displayed significant age-related attenuation of G (2) checkpoint function. Abnormalities in the function of cell cycle checkpoints were detected in transformed hepatocytes and WB-F344 cells at stages of hepatocarcinogenesis preceding tumorigenicity, sustaining a hypothesis that aberrant checkpoint function contributes to carcinogenesis.

Androgen receptor regulation of G1 cyclin and cyclin-dependent kinase function in the CWR22 human prostate cancer xenograft.

Human prostate cancer is initially dependent on androgens for growth, and androgen-dependent cells undergo apoptosis after castration. However, a subset of androgen-responsive cells survives and eventually proliferates in the absence of testicular androgen. The high levels of androgen receptor in both androgen-dependent and recurrent tumors led us to investigate androgen regulation of cell cycle proteins in human prostate cancer using the CWR22 xenograft. Cellular proliferation decreased dramatically in CWR22 tumors after castration. Testosterone propionate (TP) treatment of castrated mice restored cellular proliferation after 24-48 hours. Growth of CWR22 tumors in the absence of testicular androgen recurred several months after castration. CDK1 and CDK2, and cyclin A and cyclin B1 messenger RNAs were decreased 6 days after castration, increased 6-12 hours after TP treatment, and were expressed at high levels in recurrent CWR22 tumors. Coimmunoprecipitated cyclin B1/CDK1 and cyclin D1/CDK4 protein complexes decreased after castration and increased after TP treatment of castrated mice. In addition, CDK1 and CDK2 kinase activities were upregulated by androgen in parallel with hyperphosphorylation of retinoblastoma (Rb) protein. Despite the absence of testicular androgen in recurrent CWR22, the levels of these androgen-regulated cyclin/ CDK protein complexes and hyperphosphorylation of Rb were equal to or greater than in tumors from intact mice. The results indicate that androgen receptor regulates cellular proliferation by control of CDK and cyclins at the transcriptional level and by post-translational modifications that influence cell cycle protein activity.

Telomere shortening, telomerase expression, and chromosome instability in rat hepatic epithelial stem-like cells.

Telomeres, which are specialized structures consisting of T2AG3 repeats and proteins at the ends of chromosomes, may be essential for genomic stability. To test whether telomere length maintenance preserves genomic stability in rats (Rattus rattus and Fischer 344), we assayed telomerase activity and telomere length in the rat hepatic epithelial stem-like cell line WB-F344 during aging in vitro and in tumor-derived lines. Telomerase activity in the parental WB-F344 line was repressed at low and intermediate passage levels in vitro and reexpressed at high passages. Southern blot hybridization and quantitative fluorescence in situ hybridization analyses demonstrated that telomeres were significantly eroded at intermediate passage levels, when telomerase was repressed, and at high passage levels, when telomerase was expressed. Fluorescence in situ hybridization analysis also revealed interstitial telomeric sequences in rat chromosomes. Tumor-derived WB-F344 cell lines that express telomerase had variably shortened telomeres. Cytogenetic analyses performed on WB-F344 cells at low, intermediate, and high passages demonstrated that chromosome instability was most severe in the intermediate passage cells. These data suggest that telomere shortening during aging of rat hepatic epithelial stem-like WB-F344 cells in vitro and during selection of tumorigenic lines in vivo may destabilize chromosomes. Expression of telomerase in high passage cells appeared to partially stabilize chromosomes.

Induction of rat WT1 gene expression correlates with human chromosome 11p11.2-p12-mediated suppression of tumorigenicity in rat liver epithelial tumor cell lines.

We have previously identified and mapped a locus within human chromosome 11p11.2-p12 that suppresses the tumorigenic potential of some rat liver tumor cell lines. In the present study, possible molecular mechanisms of human 11p11.2-p12-mediated liver tumor suppression were investigated by examining gene expression patterns in suppressed and non-suppressed microcell hybrid (MCH) cell lines. The parental rat liver tumor cell lines (GN6TF and GP7TB) express moderate levels of p53 mRNA and protein, overexpress mRNAs for c-H-ras, c-myc, and TGFá, and do not express detectable levels of WT1 mRNA or protein. Suppression of tumorigenicity by human chromosome 11p11.2-p12 was not accompanied by significant alterations in the levels of expression of p53, c-myc, or TGFá. Expression of c-H-ras was decreased significantly in both suppressed and non-suppressed MCH cell lines, suggesting that down-regulation of c-H-ras is not directly responsible for tumor suppression. In contrast, the level of expression of WT1 correlated precisely with tumor suppression in this model system. All suppressed MCH cell lines expressed WT1 mRNA and protein at levels comparable to that of untransformed rat liver epithelial cells (WB-F344), whereas only trace WT1 mRNA and protein were detected in a non-suppressed MCH cell line. PCR analysis demonstrated that two suppressed MCH cell lines do not carry the human WT1 gene, indicating that WT1 expression in these lines originates from the rat locus. Furthermore, RT-PCR analysis showed that each of the four known splice variants of the WT1 mRNA are expressed in these suppressed MCH cell lines, recapitulating the expression pattern observed in the untransformed rat liver epithelial cells. Re-expression of tumorigenicity by suppressed MCH cell lines was accompanied by the coordinate loss of human chromosome 11p11.2-p12 and of WT1 gene expression, suggesting that one or more human 11p11.2-p12 genes are required for sustained expression of WT1 in these cell lines. Together, these results suggest that the molecular mechanism governing human chromosome 11p11.2-p12-mediated liver tumor suppression may involve induction of rat WT1 gene expression under the direct or indirect transcriptional regulation of a genetic locus (or loci) on human 11p11.2-p12.

Isolation and partial characterization of an epithelial cell line (RPE-F344) from the regenerating prostate of a normal adult male rat.

Normal prostate epithelial cells are difficult to propagate in vitro without experimental immortalization. The goal of this study was to isolate and characterize a propagable epithelial cell line from normal adult rat prostate. Enrichment of proliferation-competent cells was accomplished in vivo by initiating a single cycle of prostatic involution/regeneration. The RPE-F344 cell line was established from an androgen-deprived, involuted prostate four days after the initiation of regeneration by administration of testosterone. The cell line has been cultured in vitro for >50 passages, forms a uniform monolayer in culture, exhibits contact inhibition at confluence, and does not form colonies in soft agar. Immunocytochemical and RT-PCR analyses demonstrated that the RPE-F344 cells express anti-apoptotic genes associated with cell survival, and several growth factor receptors important in prostate development and homeostasis. RPE-F344 cells are p27kip1 negative, telomerase positive, and express high molecular weight cytokeratins specific for prostatic basal cells. They also express low levels of androgen receptor (AR) and prostatic acid phosphatase (PAP); features associated with secretory luminal epithelial cells. RPE-F344 cells are maintained in vitro without androgen supplementation, but addition of 15nM dihydrotesterone (DHT) to the culture media results in a significant but transient enhancement of cellular proliferation. Establishment of RPE-F344-like colonies from rat prostate is limited to the ventral and dorsal lobes of the prostate 2-4 days after initiation of regeneration, suggesting that RPE-F344 cells may originate from a stem cell-like compartment that is responsible for regenerative repopulation.

Spontaneous neoplastic transformation of WB-F344 rat liver epithelial cells.

Several studies have shown that cultured rat liver epithelial cells transform spontaneously after chronic maintenance in a confluent state in vitro. In the present study, multiple independent lineages of low-passage WB-F344 rat liver epithelial stem-like cells were initiated and subjected in parallel to selection for spontaneous transformation to determine whether spontaneous acquisition of tumorigenicity was the result of events (genetic or epigenetic) that occurred independently and stochastically, or reflected the expression of a pre-existing alteration within the parental WB-F344 cell line. Temporal analysis of the spontaneous acquisition of tumorigenicity by WB-F344 cells demonstrated lineage-specific differences in the time of first expression of the tumorigenic phenotype, frequencies and latencies of tumor formation, and tumor differentiations. Although spontaneously transformed WB-F344 cells produced diverse tumor types (including hepatocellular carcinomas, cholangiocarcinomas, hepatoblastomas, and osteogenic sarcomas), individual lineages yielded tumors with consistent and specific patterns of differentiation. These results provide substantial evidence that the stochastic accumulation of independent transforming events during the selection regimen in vitro were responsible for spontaneous neoplastic transformation of WB-F344 cells. Furthermore, cell lineage commitment to a specific differentiation program was stable with time in culture and with site of transplantation. This is the first report of a cohort of related, but independent, rat liver epithelial cell lines that collectively produce a spectrum of tumor types but individually reproduce a specific tumor type. These cell lines will provide valuable reagents for investigation of the molecular mechanisms involved in the differentiation of hepatic stem-like cells and for examination of potential causal relationships in spontaneously transformed rat liver epithelial cell lines between molecular/cellular alterations and the ability to produce tumors in syngeneic animals.

Establishment of a functional HGF/C-MET autocrine loop in spontaneous transformants of WB-F344 rat liver stem-like cells.

A model of spontaneous malignant transformation was used to evaluate the molecular changes that take place in WB-F344 rat liver epithelial cells during neoplastic transformation and tumorigenesis. A comparison of wild-type low-passage WB-F344 cells to spontaneously transformed tumor cell lines revealed that the majority of the tumor cell lines have an increased capacity for autonomous proliferation and motility when maintained in serum-free media. In the current study, we show that c-met is expressed at some level in wild-type WB-F344 cells and in all of the spontaneously transformed tumor cell lines, and that 9/16 of the tumor cell lines have acquired hepatocyte growth factor (HGF) expression. In vitro growth of HGF-expressing tumor cell lines is inhibited as much as 68% by the addition of neutralizing antibodies to HGF or antisense HGF oligonucleotides, indicating that the production of HGF by the tumor cells is partially responsible for driving autonomous proliferation in a subset of tumor cell lines. Furthermore, conditioned media collected from HGF-expressing tumor cell lines stimulates DNA synthesis in wild-type WB-F344 cells, and this effect can be abrogated by pre-incubation of the conditioned media with neutralizing antibodies to HGF. Because HGF is a motility-promoting growth factor, all cell lines were evaluated to determine if expression of HGF stimulated motogenesis. All tumor cell lines (regardless of HGF expression) were highly motile in comparison with wild-type WB-F344 cells, with a 3.5-fold to 20-fold greater number of motile cells. The high basal rate of motility characteristic of the tumor cell lines is not a result of the production of HGF, because it is also a property of the cell lines that do not express HGF messenger RNA. Furthermore, tumor cell motility is not inhibited by antisense oligonucleotides or neutralizing antibodies. Establishment of an autocrine HGF/c-met loop in a subset of spontaneously transformed WB-F344 cell lines may influence development and/or expression of the tumorigenic phenotype by driving cellular proliferation.

Genomic fluidity is a necessary event preceding the acquisition of tumorigenicity during spontaneous neoplastic transformation of WB-F344 rat liver epithelial cells.

The genomic evolution of a cohort of WB-F344 rat liver epithelial cell lineages undergoing spontaneous neoplastic transformation was followed to define the mechanistic relationship between genomic instability and progression to the neoplastic phenotype. Eighteen independent populations of WB-F344 cells (initiated from a single diploid-founding population) were subjected to 12 cycles of selective growth at confluent cell density, and cellular DNA contents were measured after each selection cycle. Flow cytometry demonstrated significant gains in the amount of G1 DNA after selection cycles 3, 6, and 7 in 44% (8 of 18), 89% (16 of 18), and 39% (7 of 18) of the cell populations, respectively. All populations subsequently lost DNA and returned to a diploid or pseudo-diploid DNA content within 1 to 2 selection cycles after the appearance of an increased DNA content. Additionally, appearance and subsequent disappearance of aneuploid or tetraploid subpopulations was observed in 11% (2 of 18) and 83% (15 of 18) of the experimental lineages, respectively. Although perturbations of G1 DNA content were apparent as early as selection cycle 3, at least 8 cycles of selective growth were required for the acquisition of tumorigenicity. While the independent lineages demonstrated significant fluctuations in G1 DNA content between selection cycles 3 and 8, the majority (11 of 13) of the populations contained a diploid or pseudo-diploid DNA content at the time tumorigenicity was expressed. Genomic instability preceded the acquisition of tumorigenic potential in rat liver epithelial cells subjected to selective growth conditions of maintenance at confluence, and may be required for its expression.

Isolation and characterization of propagable cell lines (HUNC) from the androgen-sensitive Dunning R3327H rat prostatic adenocarcinoma.

The Dunning H rat prostate tumor (R3327H) is a widely used experimental model of human prostatic adenocarcinoma (CaP). The Dunning H tumor has been characterized as androgen-sensitive, androgen-receptor (AR) positive, prostate-specific antigen and prostatic acid phosphatase (PAP) positive. To date, the tumor has been maintained by serial passage in vivo because of the lack of an in vitro cell line that retains the characteristics of the in vivo tumor. The objective of the present study was to establish a propagable cell line from R3327H adenocarcinoma that maintained androgen sensitivity and expression of AR, PSA and PAP. Tissue harvested from an in vivo R3327H tumor was dissociated with collagenase and placed into Richter's improved media (with supplements). A cytokeratin-positive epithelial cell line (HUNC-E) and a vimentin-positive stromal cell line (HUNC-S) were generated from the primary culture, subcultured continuously for >300 days, and passaged >50 times. Survival of the HUNC-E cell line in vitro depended on several media supplements, including nicotinamide, insulin, transferrin, selenium and epidermal growth factor (EGF). HUNC-E cells expressed AR and produced PSA and PAP throughout the culture period, as confirmed by immunocytochemistry and Western blot analyses. Addition of 14 nM testosterone (T) or dihydrotestosterone (DHT) to HUNC-E cells, stimulated DNA synthesis as well as anchorage-independent growth and PSA production, which demonstrated the androgen-sensitive nature of the cells in vitro. When HUNC-E and HUNC-S cells were combined in a 3:1 ratio and introduced subcutaneously into syngeneic male hosts, tumors formed in 2/3 animals with an average latency of 7 months. RT-PCR and immunocytochemical characterization of the HUNC cell lines revealed that the cells expressed several growth factors and their cognate receptors, including HGF, TGF-alpha and the TGF-betas, indicating the establishment of potential autocrine loops in the neoplastic cells. The HUNC-E and HUNC-S CaP cell lines, which retain the characteristics of the epithelial and stromal components of the in vivo R3327H tumor, will allow a more thorough and informative molecular and biological analysis of prostatic adenocarcinoma.

Failure to achieve gene conversion with chimeric circular oligonucleotides: potentially misleading PCR artifacts observed.

Recently, a novel strategy for nucleotide exchange of target DNA using chimeric RNA/DNA oligonucleotides (CO) was reported. The CO can easily be transfected into cells, remain stable within the cells, and migrate to the nucleus. We have in this study used 42 similar constructs for targeting six different human and canine loci. A variety of cationic lipids, electroporation, and microinjection were used for transfection of the CO into lymphoblastoids, Huh7, HT 1080, and Jurkat cell lines, and canine primary fibroblasts and hepatocytes. However, no nucleotide exchange was detected in any of the targeted loci. Using PCR followed by restriction enzyme analysis, nucleotide exchange in approximately 2%-10% of the PCR products was observed during the first 3 days after transfection with CO-vWF-28S2 designed for repairing a mutation in the von Willebrand gene. Surprisingly, the observed exchange reverted after culturing the cells for a longer period of time (14 days). Furthermore, a positive indication of gene conversion (5%) was also obtained using an allele-specific PCR method for analysis of the PAI-1 gene. However, cloning of the PCR products revealed no nucleotide exchange. In our view, the most likely explanation is that the initial false positive result originates from a PCR artifact created by the CO itself. Our results imply that an independent method, that is, Southern blotting, must be used to verify an observed nucleotide exchange.

Expression of telomerase in normal and malignant rat hepatic epithelia.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA repeats onto the ends of chromosomes. More than 85% of human cancers express telomerase activity and a large proportion of human hepatocellular carcinomas are positive. To study the role of telomerase expression in rat hepatocarcinogenesis, telomerase activity was assayed in various rat tissues and in two types of liver epithelial cells: hepatocytes and hepatic epithelial stem-like cells. In the present study, we demonstrate that telomerase activity in rats is tissue-specific and stable with animal aging. Liver and testis were found to be telomerase positive, spleen had low or no activity, and kidney was negative. Telomerase activity did not change significantly in 18 month-old rats compared to 2 month-old rats, but was moderately (twofold) increased during liver regeneration induced by a 2/3's partial hepatectomy. Telomerase activity was detected in isolated rat hepatocytes and low passage hepatic epithelial stem-like cells (WB-F344). Telomerase activity displayed significant variations in a propagable clone of WB-F344 cells. At low passage levels after establishment in vitro (passages 4-9) non-tumorigenic WB-F344 cells expressed telomerase activity. During further in vitro passaging these cells lost expression of telomerase. Expression of telomerase in the tumor-derived lines of WB-F344 cells but not in the selectively cycled, parental lineages of these cells suggests that there may be a role for telomerase in hepatocarcinogenesis.

Modifications of the hepatocyte growth factor/c-met pathway by constitutive expression of transforming growth factor-alpha in rat liver epithelial cells.

We have previously shown that rat liver epithelial cells (RLEC) transfected with and constitutively expressing transforming growth factor-alpha (TGF-alpha) have an enhanced mitogenic response to hepatocyte growth factor (HGF). In the study reported here, we examined tumor clones derived from the TGF-alpha transfectants with respect to mitogenic response to HGF. Tumor cell lines that expressed TGF-alpha responded to HGF with a greater increase in DNA synthesis than did the nontransfected parental RLEC (pRLEC). The tumor clones had also acquired a lower threshold for HGF response, which enabled them to undergo significant DNA synthesis at a low concentration of HGF that did not evoke a response in the pRLEC or TGF-alpha transfectants. We investigated the mechanisms by which TGF-alpha expression may influence the HGF/c-met pathway. We showed that most TGF-alpha transfectants and tumor cells displayed increases in c-met mRNA and protein, indicating that the enhanced HGF response may be due in part to an increase in the amount of receptor present. However, in all transfectants and tumor clones that constitutively expressed TGF-alpha, c-met was tyrosine phosphorylated in the absence of ligand (HGF) or other exogenous growth factors. These data suggest that induction of c-met mRNA and transactivation of c-met may be a sequela of the constitutive expression of TGF-alpha and that constitutive activation of the epidermal growth factor receptor pathway leads to phosphorylation and activation of c-met. These studies provide evidence for a novel mechanism of communication between epidermal growth factor receptor and c-met pathways that may partially explain the synergistic effects reported between TGF-alpha and HGF.

Investigation of the cooperative effects of transforming growth factor alpha and c-myc overexpression in rat liver epithelial cells.

Overexpression of both transforming growth factor (TGF)-alpha and c-myc is consistently reported in hepatic tumors. We transfected rat liver epithelial cells (RLECs) with expression vectors for TGF-alpha, c-myc, or both and analyzed the morphology, biological properties, and tumorigenicity of clones that overexpressed these genes. The transfectants were morphologically indistinguishable from the parental RLECs, but the overexpression of TGF-alpha resulted in changes in growth properties and an enhanced response to the mitogenic effects of hepatocyte growth factor. The concomitant overexpression of c-myc decreased growth factor requirements of the TGF-alpha lc-myc clones compared with RLEC and TGF-alpha clones. The TGF-alpha and TGF-alpha lc-myc clones were tumorigenic in nude mice at frequencies of 27% and 53%, respectively, indicating that the genes cooperate in malignant transformation. However, the untransformed nature and low tumorigenicity of the transfectants suggest that transformation depends on other cellular events in addition to the overexpression of TGF-alpha or c-myc. Characterization of tumor cell lines showed that in contrast to the transfectants, the tumor clones were morphologically transformed, capable of autonomous growth and anchorage-independent growth, and aggressively tumorigenic with a frequency of 100%. Clearly, the tumor cells differed from the transfectants and had undergone biological or genetic alterations (or both) as a consequence of the overexpression of TGF-alpha or c-myc. Our data suggest that the overexpression of TGF-alpha leads to enhanced responsiveness to hepatocyte growth factor, whereas the concomitant overexpression of c-myc confers growth-factor independence, providing a potential explanation of the mechanisms by which the overexpression of these genes results in transformation.